The advent of recombinant DNA technology has led to substantial effort to develop methods to facilitate the transfection and transduction of therapeutic and other nucleic acid-based agents to specific cells and tissues. Known techniques provide for the delivery of such agents with a variety of genes, provided in recombinant expression constructs. These constructs are capable of mediating functionality of the genes once they arrive within a cell. Such developments have been critical to many forms of molecular medicine, specifically gene therapy, whereby a missing or defective gene can be replaced by an exogenous copy of the functional gene.
Introduction of foreign nucleic acid into a cell can be accomplished chemically, biologically, or mechanically. Some current methods include viral transduction and non-viral delivery, such as electroporation, lipid dependent, polymer dependent, polypeptide dependent delivery, calcium co-precipitation and transfection with “naked” DNA.
Viral approaches typically use a genetically engineered virus to infect a host cell, thereby “transducing” the cell with an exogenous nucleic acid. Among known viral vectors are recombinant viruses, poxviruses, herpes viruses, adenoviruses, and retroviruses. Such recombinants can carry heterologous genes under the control of promoters or enhancer elements, and are able to cause their expression in vector-infected host cells, as reviewed in Mackett et al., J. Virol. 49:3 (1994); Kotani et al., Hum. Gene Ther. 5:19-28 (1994). However, viral transfection approaches carry a risk of mutagenicity due to viral integration into the cellular genome, or as a result of undesirable viral propagation. Insertion of retroviral DNA can result in inactivation or ectopic activation of cellular genes, thereby causing diseases (Lee et al., J. Virol. 64:5958-5965 (1990)) or activation of oncogenes (Shiramizu et al., Cancer Res., 54:2069-2072 (1994)). Furthermore, viral vectors also are susceptible to undesirable interactions with the host immune system.
Non-viral methods of gene delivery include such as electroporation, liposomal, polymer, polypeptide dependent delivery and transfection with “naked” DNA. Electroporation utilizes the application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of pores and other disturbances in the plasma membrane (U.S. Pat. No. 4,394,448 to Szoka, Jr., et al. and U.S. Pat. No. 4,619,794 to Hauser). Nucleic acids can enter directly into the cell cytoplasm either through these as a consequence of the redistribution of membrane components that accompanies closure of the pores and membrane restoration. Liposomal and polypeptide dependent approaches mix the material to be transferred with lipids or non-toxic polymers to form particles able to penetrate cells and to deliver nucleic acisd into cytoplasm (Felgner and Ringold, Nature, 337:387-388 (1989), Saltzman and Desai, Annals of Biomedical Engineering, 34, 270-275 (2006).
Polypeptide dependent approaches involve the use of highly penetrating proteins and peptides mixed with a nucleic acid followed by exposure of a target cell to the nucleoprotein/nucleopeptide complex (Verma and Somia, Nature, 389:239 (1997); Wolff et al., Science, 247:1465 (1990)). “Naked” DNA transfection approaches involve methods where nucleic acids are administered directly in vivo, for example, as described in U.S. Pat. No. 5,837,693 to German et al. In the “Naked” DNA approach, the nucleic acid is injected or otherwise contacted with the cells without any adjuvants.
A common disadvantage to known non-viral DNA delivery techniques is that the amount of exogenous protein expression produced relative to the amount of exogenous nucleic acid administered remains too low for most diagnostic or therapeutic procedures. Low levels of protein expression are often a result of a low rate of transfection of the nucleic acid and/or toxicity of exogenous DNA. In addition, some types of cells are very resistant to DNA transfection, and introduced foreign DNA can incorporate into the genome and act as a mutagen.
An alternative procedure for non-viral gene delivery is achieved by transfection of mRNA rather than DNA. In principle, unlike DNA transfection, introducing mRNA can have no permanent effect on the genetic structure of the cell, at least in the absence of rare reverse transcription events. There is limited literature on the application of mRNA transfection approaches (for example, Seaboe-Larssen, et al., J. Imm. Methods, 259:191-203 (2002); Boczkowski et al., Cancer Res., 60:1028-1034 (2001); and Elango et al., Biochem. Biophys. Res. Comm., 330, 958-966 (2005)), and little in the way of a systematic comparison of DNA and RNA transfection procedures. Most available literature for mRNA transfection is based on methods that involve labor intensive cloning the gene of interest in special vectors containing a bacteriophage upstream and polyA/T stretch downstream of the cloning site. Not only is cloning time consuming, but recombinant plasmids containing a stretch of poly(A/T) are often unstable in bacterial cells and prone to spontaneous mutations (Kiyama, et al., Gene, 150:1963-1969 (1994). Furthermore, most mRNAs that are generated from d(A/T)n vectors contain a short sequence of heterologous nucleotides following the poly(A) tail. The influence of these heterologous sequences on translation is unknown (Elango et al., Biochem. Biophys. Res. Comm., 330, 958-966 (2005). There is therefore a need for a transfection method that circumvents the problems associated with vector-dependent transfection methods.
It is an object of the present invention to provide a more convenient and/or efficient method of mRNA production for transfection of different types of cells, including the types which are not transfectable for DNA.
It is also an object of the present invention to provide a method of mRNA transfection with minimal side effects and high efficiency, which allows transient expression of genes and desirable modification of cell phenotype without cause permanent genetic changes, which avoids risk associated with conventional gene therapy
It is also an object of the present invention to provide a method of cell transfection with multiple genes wherein the level of each gene expression can be individually controlled.
It is also an object of the present invention to provide a method of transfection of primary mammalian cells, including human cells, and use those cells for therapy of cancer, autoimmune and infectious diseases.
It is another object of the present invention to provide a method of transient cell modification, which allows fast and safe generation of diverse differentiation states of cells of different cell types, including diverse stem cells from various tissues such as fibroblasts, hematopoietic, epithelial cells and others.